Francisella tularensis, the causative agent of tularemia, modulates the host immune response to gain a survival advantage within the host. One mechanism of immune evasion is the ability of F. tularensis to induce the synthesis of the small lipid mediator prostaglandin E2 (PGE 2 ), which alters the host T cell response making the host more susceptible to Francisella growth. PGE 2 is synthesized by a tightly regulated biosynthetic pathway following stimulation. The synthesis of PGE 2 begins with the liberation of arachidonic acid (AA) from membrane phospholipids by cytosolic phospholipase A2 (cPLA 2 ). AA is subsequently converted to the unstable intermediate PGH 2 by cyclooxygenase-2 (COX-2), and PGH 2 undergoes an isomerization reaction to generate PGE 2 . Our objective was to identify F. tularensis-activated host signaling pathways that regulate the activity of the enzymes in the PGE 2 -biosynthetic pathway. In this study, we show that cPLA 2 , p38 mitogen-activated protein kinase (MAPK), and Janus kinase 3 (JAK3) signaling are necessary for F. tularensis-induced PGE 2 production. Inhibition of JAK3 activity reduced the phosphorylation of cPLA 2 and COX-2 protein levels. In addition, JAK3 regulates cPLA 2 phosphorylation independent of transcription. Moreover, p38 MAPK activity is required for F. tularensis-induced COX-2 protein synthesis, but not for the phosphorylation of cPLA 2 . This research highlights a unique signaling axis in which JAK3 and p38 MAPK regulate the activity of multiple enzymes of the PGE 2 -biosynthetic pathway in macrophages infected with F. tularensis.
Francisella tularensis induces the synthesis of prostaglandin E 2 (PGE 2 ) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE 2 synthesis by F. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA 2 ), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE 2 synthesis, cPLA 2 hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE 2 . However, in F. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA 2 phosphorylation (a marker of activation) and PGE 2 synthesis. These results suggested to us that cPLA 2 is not responsible for the liberation of AA to be converted into PGE 2 by F. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE 2 biosynthesis. cPLA 2 , on the other hand, was required for macrophage cytokine responses to F. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response to F. tularensis. Our results identify both an alternative pathway for inducible PGE 2 synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.
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